Citations

Recent studies highlight transcriptionally distinct fibroblast subpopulations in human lungs. We observed the loss of these native transcriptional programs as fresh isolated cells are maintained in traditional culture conditions. Identifying the signals defining native fibroblast identities will be pivotal to creating culture models that preserve unique subpopulations. The screening system developed here will allow the investigation of a broad selection of cues, leading to better culture models for studying human lung fibroblast function and plasticity.

Background: Cardiac fibrosis is a critical and independent driver of heart failure (HF), characterized by excessive extracellular matrix (ECM) deposition. Decreased CDCP1 (CUB domain-containing protein 1) expression has been associated with myocardial recovery in human genome wide association studies by attenuating PDGF-BB driven cardiac fibroblast proliferation with no effect on canonical TGF-beta mediated transdifferentiation in-vitro. In-vivo, Cdcp1 deletion significantly reduced cardiac fibrosis and dysfunction in an angiotensin II/phenylephrine-induced pressure overload mouse model. However, its upstream regulators and its downstream effects on molecular signaling pathways that modulate ECM remodeling are unknown. Hypothesis: CDCP1 promotes human cardiac fibroblast mediated ECM remodeling in the HF microenvironment. Methods: Human ventricular fibroblasts (HVF) were treated with PDGF-BB (20 ng/ml), TGF-β (10 ng/ml), or Angiotensin II (100 ng/ml) for 48 hours. CDCP1 knockdown (KD) was achieved through siRNA transfection. Gene expression analysis was performed using qRT-PCR and protein quantification by Western blot. Both intact HVF and decellularized ECM were immunostained for collagen 1 to evaluate for ECM compositional changes. Results: Individual and combination treatments with fibrotic stimuli significantly increased CDCP1 expression (Figure 1A-1B). CDCP1 KD resulted in reduction of key ECM markers COL1A1, CTGF, and LOX (Figure 1C) across all fibrotic stimuli . Immunostaining of HVF after treatment with fibrotic stimuli showed reduced collagen 1 production with CDCP1 KD (Figure 1D). Western blot analysis showed increased phosphorylation of Src (tyr416) with 6h of treatment with fibrotic stimuli which was nullified by silencing of CDCP1 (Figure 1E). Conclusion: Our findings demonstrate that CDCP1 is necessary for ECM synthesis and Src signaling across multiple profibrotic pathways. Selective targeting of CDCP1 to modulate Src signaling pathways could provide novel therapeutic avenues for HF and fibrosis treatment.

Fibrosis in the lung is thought to be driven by epithelial cell dysfunction and aberrant cell-cell interactions. Unveiling the molecular mechanisms of cellular plasticity and cell-cell interactions is imperative to elucidating lung regenerative capacity and aberrant repair in pulmonary fibrosis. By mining publicly available RNA-Seq data sets, we identified loss of CCAAT enhancer-binding protein alpha (CEBPA) as a candidate contributor to idiopathic pulmonary fibrosis (IPF). We used conditional KO mice, scRNA-Seq, lung organoids, small-molecule inhibition, and potentially novel gene manipulation methods to investigate the role of CEBPA in lung fibrosis and repair. Long-term (6 months or more) of Cebpa loss in AT2 cells caused spontaneous fibrosis and increased susceptibility to bleomycin-induced fibrosis. Cebpa knockout (KO) in these mice significantly decreased AT2 cell numbers in the lung and reduced expression of surfactant homeostasis genes, while increasing inflammatory cell recruitment as well as upregulating S100a8/a9 in AT2 cells. In vivo treatment with an S100A8/A9 inhibitor alleviated experimental lung fibrosis. Restoring CEBPA expression in lung organoids ex vivo and during experimental lung fibrosis in vivo rescued CEBPA deficiency–mediated phenotypes. Our study establishes a direct mechanistic link between CEBPA repression, impaired AT2 cell identity, disrupted tissue homeostasis, and lung fibrosis.

AbstractPulmonary fibrosis is characterized by pathological accumulation of scar tissue in the lung parenchyma. Many of the processes that are implicated in fibrosis, including increased extracellular matrix synthesis, also occur following pneumonectomy (PNX), but PNX instead results in regenerative compensatory growth of the lung. As fibroblasts are the major cell type responsible for extracellular matrix production, we hypothesized that comparing fibroblast responses to PNX and bleomycin (BLM) would unveil key differences in the role they play during regenerative versus fibrotic lung responses. RNA‐sequencing was performed on flow‐sorted fibroblasts freshly isolated from mouse lungs 14 days after BLM, PNX, or sham controls. RNA‐sequencing analysis revealed highly similar biological processes to be involved in fibroblast responses to both BLM and PNX, including TGF‐β1 and TNF‐α. Interestingly, we observed smaller changes in gene expression after PNX than BLM at Day 14, suggesting that the fibroblast response to PNX may be muted by expression of transcripts that moderate pro‐fibrotic pathways. Itpkc, encoding inositol triphosphate kinase C, was a gene uniquely up‐regulated by PNX and not BLM. ITPKC overexpression in lung fibroblasts antagonized the pro‐fibrotic effect of TGF‐β1. RNA‐sequencing analysis has identified considerable overlap in transcriptional changes between fibroblasts following PNX and those overexpressing ITPKC.

AbstractPulmonary arterial hypertension (PAH) causes pulmonary vascular remodeling, increasing pulmonary vascular resistance (PVR) and leading to right heart failure and death. Matrix stiffening early in the disease promotes remodeling in pulmonary artery smooth muscle cells (PASMCs), contributing to PAH pathogenesis. Our research identified YAP and TAZ as key drivers of the mechanobiological feedback loop in PASMCs, suggesting targeting them could mitigate remodeling. However, YAP/TAZ are ubiquitously expressed and carry out diverse functions, necessitating a cell‐specific approach. Our previous work demonstrated that targeting non‐canonical IKB kinase TBK1 reduced YAP/TAZ activation in human lung fibroblasts. Here, we investigate non‐canonical IKB kinases TBK1 and IKKε in pulmonary hypertension (PH) and their potential to modulate PASMC pathogenic remodeling by regulating YAP/TAZ. We show that TBK1 and IKKε are activated in PASMCs in a rat PH model. Inflammatory cytokines, elevated in PAH, activate these kinases in human PASMCs. Inhibiting TBK1/IKKε expression/activity significantly reduces PAH‐associated PASMC remodeling, with longer‐lasting effects on YAP/TAZ than treprostinil, an approved PAH therapy. These results show that non‐canonical IKB kinases regulate YAP/TAZ in PASMCs and may offer a novel approach for reducing vascular remodeling in PAH.

Despite recent therapeutic developments, pulmonary fibrosis remains a debilitating disease with poor prognosis. In order to create effective treatments, promising molecular targets need to be identified and validated. Across multiple organs, mesenchymal derived myofibroblasts play an essential role in matrix deposition and tissue contraction. We previously carried out a high‐content, RNAi screen to characterize myofibroblast cytoskeletal phenotypes (α‐SMA and organized F‐actin) in cultured fibroblasts. Hits from this screen, including STAT3 (Signal transducer and activator of transcription 3), were validated in primary lung fibroblasts from patients with IPF (idiopathic pulmonary fibrosis) on compliant hydrogels. To further evaluate STAT3 as a therapeutic target, we have focused on LLL12, a small molecule inhibitor of STAT3. LLL12 dose‐dependently (0.3, 1.0μM) reduced RNA expression of profibrotic genes ( ACTA2 , CTGF , COL1A1 , and FN1 ) as well as protein deposition of collagen III and fibronectin, in TGFβ stimulated cultured fibroblasts. LLL12 also reduced contractile force, as measured by traction force microscopy, to levels comparable to that seen with inhibition of Rho kinase (Y27632, 20μM). In an effort to support the role of STAT3 as a multi‐organ regulator of tissue fibrosis we confirmed similar effects of LLL12 in primary human hepatic stellate cells and cardiac fibroblasts. Using the bleomycin model of pulmonary fibrosis we assessed the in vivo efficacy of STAT3 inhibition. The lungs of the mice treated therapeutically (day 14–28) with LLL12 (5 mg/kg i.p. daily) showed reduced collagen and fibrotic matrix content measured by hydroxyproline and trichrome histology. RNA expression of COL1A1 , FN1 , CTGF , and ACTA2 also decreased (~50%) in the mice treated with LLL12. Importantly, in the mice which did not receive bleomycin, LLL12 had no effect on RNA expression of these genes. These data not only validate the RNAi screening approach, but also point towards STAT3 as an important molecular target for regulation of myofibroblast activation in pulmonary fibrosis and other fibrotic diseases. Support or Funding Information Supported by NIH RO1 HL092961 and HL113796, and Boehringer Ingelheim.

Abstract Background Idiopathic pulmonary fibrosis (IPF) is characterized by pathological accumulation of scar tissue in the lung parenchyma. Many of the processes that are implicated in fibrosis, such as matrix deposition also occur following pneumonectomy (PNX). However, fibrosis does not occur. Since fibroblasts are the major cell type responsible for extracellular matrix production, we hypothesized that comparing their responses to PNX and bleomycin (BLM) would unveil key differences in the role they play during regulated and fibrotic lung growth. Methods RNA-sequencing was performed on flow cytometry-sorted fibroblasts from mouse lungs 14 days after bleomycin challenge, PNX, or sham. Pathway and transcription factor binding motif enrichment analysis were performed to characterize RNA-sequencing data. Normal human lung fibroblasts (NHLFs) were used as a cell culture model to validate targets. Results RNA-sequencing analysis revealed similar biological processes to be involved in both responses, including signaling by transforming growth factor-β (TGF-β1) and tumor necrosis factor-α. Transcription factor binding motif enrichment predicted erythroid transformation specific (ETS) superfamily members to play a key role in the response to BLM, whereas nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) were predicted to orchestrate much of the response to PNX. Itpkc, encoding inositol triphosphate kinase C, was a gene uniquely up-regulated by PNX and a likely AP-1 target. ITPKC overexpression in NHLFs antagonized the fibrotic effect of TGF-β1. RNA-sequencing analysis of primary NHLFs overexpressing ITPKC further supported its role in repressing collagen production and predicted it as an activator of canonical NF-κB signaling. ITPKC overexpression showed considerable overlap with the innate immune signaling seen following PNX. Conclusion Taken together, our RNA-sequencing analysis suggests that during post-PNX lung growth, AP-1 activates ITPKC to promote canonical NF-κB signaling to prevent fibrogenesis. Future studies to understand this lack of ITPKC induction during fibrotic injury may identify attractive therapeutic targets.

Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcription cofactors implicated in the contractile and profibrotic activation of fibroblasts. Fibroblast contractile function is important in alveologenesis and in lung wound healing and fibrosis. As paralogs, YAP and TAZ may have independent or redundant roles in regulating transcriptional programs and contractile function. Using IMR-90 lung fibroblasts, microarray analysis, and traction microscopy, we tested whether independent YAP or TAZ knockdown alone was sufficient to limit transcriptional activation and contraction in vitro. Our results demonstrate limited effects of knockdown of either YAP or TAZ alone, with more robust transcriptional and functional effects observed with combined knockdown, consistent with cooperation or redundancy of YAP and TAZ in transforming growth factor β1 (TGFβ1)-induced fibroblast activation and contractile force generation. The transcriptional responses to combined YAP/TAZ knockdown were focused on a relatively small subset of genes with prominent overrepresentation of genes implicated in contraction and migration. To explore potential disease relevance of our findings, we tested primary human lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis and confirmed that YAP and TAZ combined knockdown reduced the expression of three cytoskeletal genes, ACTA2, CNN1, and TAGLN. We then compared the contribution of these genes, along with YAP and TAZ, to contractile function. Combined knockdown targeting YAP/TAZ was more effective than targeting any of the individual cytoskeletal genes in reducing contractile function. Together, our results demonstrate that YAP and TAZ combine to regulate a multigene program that is essential to fibroblast contractile function.